The major long-term goal of this project is to comprehensively identify and analyze all evolutionary mutations that accumulated in five experimentally-evolved strains of the model social bacterium Myxococcus xanthus. Each strain evolved a novel form of social behavior that will be the focus of study, namely obligate cheating, re-evolved multicellular fruiting body development, re-evolved social swarming, adaptive cheater suppression and ten-fold enhanced predatory searching. Having sequenced the genomes of these strains to a high coverage level, all accumulated mutations will be identified by genome sequence, PCR and insertion element analyses. The history of mutation appearances and subsequent frequencies will be defined for each lineage among clones from evolutionary intermediate frozen populations (Specific Aim 1). The mutations that cause the phenotypes of primary interest will be identified, and each mutation will be analyzed for its effects on evolutionary fitness and social phenotypes. The degree of epistatic interaction among sequentially incurred mutations will also be quantified. These goals will be accomplished by transfer of mutations across genomic backgrounds and corresponding population-level performance assays (Specific Aim 2). The molecular pathways and mechanisms that underlie these social adaptations will be characterized by a combination of global approaches (e.g. transcriptome and proteome analysis) and testing of focused mechanistic hypotheses based on existing knowledge of M. xanthus social genetics (Specific Aim 3). This project is highly significant because it represents a ground-breaking, DNA sequence based approach to understanding bacterial social evolution at multiple levels of biological organization. This research investigates several important themes in evolutionary biology that are also relevant to the evolution of human pathogens that rely on social traits analogous to those of M. xanthus for evolutionary success.